(1) Field of the Invention
The present invention relates to a method for the colorimetric, particularly spectrophotometric, determination of calcium in serum, particularly human blood serum, using arsenazo III as an indicator which complexes with calcium to develop a color along with a hydroxyquinoline which binds magnesium in the serum without forming a colored complex. In particular, the present invention uses an alkaline aqueous reagent which contains 8-hydroxyquinoline or preferably 8-hydroxyquinoline sulfonic acid (also known as 8-hydroxyquinoline sulfonate) which complexes with magnesium so that it is non-reactive with arsenazo III.
(2) Prior Art
Arsenazo III (ASA III) has been used as a selective ligand for the colorimetric determination of several metals (Michaylova, V. and P. Ilkova, Anal Chim Acta 53:194-198 (1971); Savvin, S. B., Talanta 8:673-685 (1961); Michaylova, V. and L. Yoroukova, Anal. Chim. Acta 68:73-82 (1974); and Savvin, S. B., Doklady Akad Nauk USSR 127:1231-1234 (1959)). However, in the clinical laboratory its most common application has been limited to the determination of serum calcium. Since ASA III was first used by Michaylova and Ilkova (Michaylova V. and P. Ilkova, Anal Chim Acta 53:194-198 (1971)), these colorimetry calcium determinations have been performed at an acidic pH because of the greater selectively by ASA III for calcium over magnesium under acidic conditions, where the latter has been shown to be a major interfering ion in serum in other calcium assays involving alkaline reactions (Connerty, H. V. and A. R. Briggs, Am J Clin Path 45:290-296 (1966)). The alkaline pH region has not been used for serum determinations of calcium even though the molar absorptivity of ASA III increases with pH with a maximum value occurring at pH 9 (Michaylova V. and P. Ilkova, Anal Chim Acta 53:194-98 (1971)).
In the past, 8-hydroxyquinoline (8HQ) has been used as a masking agent for magnesium in methylthymol blue and orthocresolphthalein complexone based calcium reagents (Connerty H. V. and A. R. Briggs, Am J Clin Path 45:290-296 (1966); and Grindler, E. M. and J. D. King Am J Clin Pathol 58:376-382 (1972)), both tests being carried out in alkaline aqueous solutions. Studies have shown that the masking of magnesium by 8HQ is not always complete in these tests. In addition, the interaction of magnesium with 8HQ can also cause high results for calcium in comparison to more definitive methods (Connerty H. V. and A. R. Briggs, Am J Clin Path 45:290-296 (1966); and Grindler, E. M. and J. D. King, Am J Clin Pathol 58:376-382 (1972)).
8HQ was originally used as analytical reagent for magnesium in procedures where magnesium 8-hydroxyquinolate was first precipitated either in ammoniacal solutions or sodium hydroxide solutions containing tartrate at alkaline pHs ranging from 9.5 to 12.6. The hydroxyquinolate precipitate was then determined colorimetrically (Willard, H. H. and H. Diehl, Advanced Quantitative Analysis, 3rd ed. New York: D. Van Nostrand (1943); and Sandell, E. B., Colorimetric Determination of Traces of Metals, 3rd ed. New York: Interscience Publishers (1959)). Under conditions that the orthocresolphthalein complexone and methylthymol blue procedures are performed, the reported precipitates of magnesium quinolate have not been found, probably because the solubility maximum of the magnesium quinolate is not exceeded by the levels of magnesium encountered in serum with these reagents.
An early study (Lamkin, E. G. and M. B. Williams, Anal Chem 37:1029-1031 (1965)) provided an indirect method for determining calcium by reacting calcium and magnesium with ASA III to get a total absorbance and then removing the calcium absorbance by binding it with [ethylene bis (oxyethylene-nitrilo)] tetraacetic acid. This test involves multiple steps. A more sensitive ASA III calcium reagent would be particularly useful in applications requiring small sample volumes such as automated instrumentation and in laboratories servicing a large neonatal or geriatric population. In addition, since it has been reported that hypertriglyceridemia can cause aberrant elevated results for serum calcium when assayed spectrophotometrically (Giacola, G. P. & Krasner, Amer. J. Technol 45 767-768 (1979)), an improved reagent that would permit the on-line measurement of calcium in samples that are turbid enough to cause the light-scatter artifacts is needed.